Molecular Beacon Aptamers for Direct and Universal Quantitation ofRecombinant Proteins from Cell LysatesXiaohong Tan,† Weijun Chen,†,‡ Shun Lu,‡ Zhi Zhu,‡ Tao Chen,‡ Guizhi Zhu,†,‡ Mingxu You,‡

and Weihong Tan*,†,‡

†Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Biologyand College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China‡Center For Research at Bio/nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics,Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida, United States

ABSTRACT: Western blot, enzyme linked immunosorbent assay(ELISA), and fluorescent fusion proteins are currently the most commonmethods for detecting recombinant proteins. However, the former two arecumbersome and time-consuming, and the latter method may interferewith the trafficking and function of the fused recombinant proteins. Wereport here a rapid, inexpensive, and simple approach to detect andquantify recombinant proteins using an anti-His-tag molecular beaconaptamer (HMBA). We demonstrated the technique by detection andquantitation of expressed recombinant proteins directly from E. coli celllysate. The amount of expressed P78-His was determined to be 1.49 μgfrom the 20 μg cell lysate proteins. To the best of our knowledge, this is the first example directly measuring the concentrationand expression yield of recombinant proteins from cell lysate, and the entire procedure required only 5 min.

Recombinant proteins are generated from expression ofrecombinant DNA within host cells. The use of

recombinant proteins has expanded greatly in the last severaldecades. A large number of antibodies, antigens, hormones, andenzymes used in molecular biology, biochemistry, and medicineare obtained from expression systems. For academic andindustrial production of recombinant proteins, a simple andrapid detection method as an early operation unit can improvethe overall process. However, current methods for detection ofrecombinant proteins have limitations. Western blot andenzyme linked immunosorbent assay (ELISA) are the tradi-tional methods to detect recombinant proteins, but they arecumbersome and time-consuming. For example, becauseexpression proteins have to be separated from other cell lysateproteins before detection, laborious procedures such as gelelectrophoresis are employed in Western blot. If recombinantproteins can be directly detected from cell lysate, the detectionassay could be largely simplified. Therefore, an alternativemethod was developed by the use of genetically fused fusionpartners, such as the fluorescent GFP-fusion protein.1 BecauseGFP is coexpressed with the target recombinant protein, theexpression level of target proteins can be determined throughthe detection of the presence of GFP. This is a rapid andstraightforward approach to detect recombinant proteins.However, the large size of the fluorescent proteins (around30 kD) can interfere with the trafficking and function of theproteins to which they are fused, limiting wide application offluorescent fusion proteins to various recombinant proteins.Smaller tags have less potential to disrupt protein folding or

function, and this inspired us to use a small fused tag to detectrecombinant proteins directly from cell lysates.Although many different fusion tag systems, such as chitin

binding protein (CBP), maltose binding protein (MBP),glutathione-S-transferase (GST), and the poly histidine tag(His-tag) are available, the first choice for most scientists is theHis-tag,2 which is an amino acid motif that usually consists ofsix histidine (His) residues at the N- or C-terminus of proteins.Because of its small size (∼1 kD), His-tag rarely interferes withthe function, activity, or structure of target proteins, andtherefore, theoretically, it can be used for all recombinantproteins. Furthermore, a small His-tag can easily be geneticallyfused to a target gene by polymerase chain reaction (PCR)techniques,3 and as a mature technology, various His-taggedproteins have been successfully expressed from diverseexpression systems, including E. coli, yeast, mammalian cells,insect cells, and plant cells.4 As a result, because eachrecombinant protein could be coexpressed with one His-tag,through the monitoring of the His-tag, the detection andquantitation of the target recombinant protein could berealized.Currently, the most common methods to detect recombinant

His-tagged proteins use anti-His-tag antibodies5−7 or probesbearing metal ions, such as Ni2+ or Co2+, which can bind to theHis-tag.2,8 However, the use of antibodies lacks accuratequantitation, is costly, and requires extensive time to obtain

Received: June 25, 2012Accepted: August 23, 2012Published: August 23, 2012

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© 2012 American Chemical Society 8272 dx.doi.org/10.1021/ac301764q | Anal. Chem. 2012, 84, 8272−8276

results. Although they are cheaper than antibodies, methodsusing metal ion probes still require several hours for detectionof His-tagged proteins and lack the ability to provide accuratequantitation. More importantly, neither method is feasible fordirect detection of His-tagged proteins from cell lysate. Toaddress all these problems, we developed a novel approach toquickly detect and quantify recombinant proteins directly fromcell lysates. This method relying on an anti-His-tag molecularbeacon aptamer is universal in scope, inexpensive, and simpleenough.Aptamers are single-stranded nucleic acids generated by

SELEX (systematic evolution of ligands by exponentialenrichment).9,10 Aptamers fold into well-defined three-dimen-sional structures that enable specific binding to a great varietyof targets, such as proteins, with high affinity and specificitycomparable to that of antibodies. Anti-His-tag aptamer, 6H5,was first reported in a U.S. patent, in which it was used as anaffinity probe to purify His-tagged proteins.11 However, sinceaptamers are much more expensive than metal ion probes,aptamer affinity chromatography is not economically feasiblefor His-tagged protein purification. Nevertheless, as powerfulprobes, anti-His-tag aptamers can be used for His-taggedprotein detection. For example, anti-His-tag aptamers could beimmobilized on a solid surface to act as a DNA microarray forHis-tagged protein detection.12 However, the otherwise wideapplication of this approach has been limited by the need toprelabel target proteins with fluorescent reporters. Ourapproach eliminates protein labeling by modifying aptamerinto a molecular beacon aptamer (MBA).An MBA, also called an aptamer switch probe13 or activatable

aptamer probe,14 is a newly developed molecular beacon whichcan specifically recognize various target molecules, such asadenosine triphosphate (ATP), proteins, or even cells.13,14

Although inspired by the earlier MBAs, the design of anti-His-tag molecular beacon aptamer (HMBA) was not easy. Thesimplest way to construct the MBA would be to keep the entirelength of 6H5 and add a short cDNA sequence at its 3-primeend with a PEG linker to form a beacon structure.Unfortunately, that MBA cannot bind to His-tag (data notshown). As shown in Figure 1A, 6H5 has a molecular beacon-like hairpin structure with two tails on both ends. Wespeculated the hairpin structure could be very important forbinding between 6H5 and His-tagged proteins, and the tailsmight not be that important. Then, we decided to cut off thefive nucleotide residues (GGCTT), one-by-one, from the 5-prime end of 6H5, and these truncated aptamers were matched,respectively, with their corresponding short cDNA sequencesand a PEG linker to form MBAs. These MBAs were tested, andone of them showed good fluorescence recovery after binding

to His-tagged proteins. We named this one HMBA. As shownin Figure 1C, the HMBA contains a major part of 6H5 with thedeletion of 5 nucleotide residues from the 5-prime end andaddition of a partial cDNA sequence containing 7 nucleotideresidues to the 3-prime end and PEG36 as a linker. Fluorescentdye 6-FAM was chosen as the fluorophore, and Dabcyl wasused as the quencher.Thus, in the absence of His-tagged proteins, HMBA forms a

stem-loop structure (Figure 1C), and the fluorescence signal ofFAM is quenched by Dabcyl. However, in the presence of His-tagged proteins, which bind to HMBA, the stem part will bereorganized to physically separate fluorophore from quencher,allowing a fluorescent signal to be emitted upon excitation(Scheme 1). The entire mechanism for the use of HMBA to

detect recombinant proteins from cell lysates is illustrated inScheme 1. The recombinant protein is fused with a His-tag.After cell lysis, the HMBA can directly detect and quantify therecombinant protein from cell lysates through the interactionwith the His-tag. For this HMBA assay, it should be noted thatthe recognition of the target protein occurs simultaneously withthe optical reporting of that interaction, which is an obviousadvantage for any homogeneous high-throughput assay.Neither anti-His-tag antibodies nor metal ion probes havethis functionality.In order to verify the feasibility of the use of the HMBA to

detect recombinant His-tagged proteins, protein Rep78 with aHis-tag on its N-terminus (P78-His) was tested as a modelstudy. P78-His was expressed and purified from E.coli cells. Asshown in Figure 2, the fluorescence of HMBA was largelyincreased in the presence of P78-His. In addition, HMBA wasmixed with its cDNA, and only a slightly stronger fluorescencesignal was observed, compared with that produced by P78-His.To explore the specificity of the HMBA assay for

recombinant His-tagged proteins, the kinetic behavior of

Figure 1. The secondary structures of (A) 6H5, (B) truncated 6H5, and (C) HMBA.

Scheme 1. HMBA Used to Detect and Quantitate ExpressedRecombinant Proteins Directly in Cell Lysate

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HMBA with P78-His or P78 was studied by monitoring thefluorescence intensity change as a function of time. As shown inFigure 3, the original fluorescence signal of 25 nM HMBA in

buffer was low and stable, but when 300 nM of P78-His wasadded to the solution, the fluorescence immediately increasedto a much higher level and continued to increase slowly as afunction of time. In contrast, HMBA showed almost noresponse to 300 nM P78. These results not only confirm thatHMBA is specific to the His-tag but also illustrate that thebinding between His-tagged protein and HMBA is rapid, withnearly 90% response being reached within 5 min.Furthermore, both protein Rep68 with a His-tag on its N-

terminus (P68-His) and P68 were tested with HMBA. Asshown in Figure 4, HMBA shows strong specificity to the His-tagged proteins. There is a distinguishable difference betweenthe original proteins (P68 and P78) and His-tagged proteins(P68-His and P78-His). In addition, bovine serum albumin(BSA), as another control protein, was tested at twoconcentrations: 200 nM and 10 μM. Even when theconcentration of BSA was 50-fold higher than that of His-tagged proteins, it was found that BSA was unable to bind andopen the HMBA. Additionally, two other His-taggedrecombinant proteins, GSTZ-His and Pdx1-His, were testedin this HMBA assay, and both displayed behavior very similarto that of P78-His (Figure 4).The HMBA assay was also tested with the His-tag itself. One

short His-tag peptide, H-His-His-His-His-His-His-NH2 (H6),

opened the HMBA and gave fluorescence recovery. Althoughthe concentration of H6 was 40-fold higher than that of His-tagged proteins, the efficiency of H6 targeting HMBA was stilllower than that of His-tagged proteins (Figure 4). This couldbe attributed to the binding between HMBA and the very shortpeptide H6, which is probably weaker than that betweenHMBA and His-tagged proteins. It may be because the localenvironment of protein surface can provide additional supportfor 6H5 to bind the small His-tag. In addition, the presence ofHis-tags in all these His-tagged proteins was verified byWestern blot analysis using anti-His-tag antibodies (Figure 5).The data presented here confirm the specificity of HMBAtoward His-tags and evaluate the potential of detection ofrecombinant proteins through monitoring the fused His-tag.Different concentrations of P78-His (6.25−400 nM) were

tested with HMBA in buffer. As shown in Figure 6, significantfluorescence emission was observed when P78-His reached 100nM. The inset of Figure 6 shows the fluorescence increase (F/F0) of HMBA upon addition of different concentrations of P78-His. The linearity of the plot indicates that the HMBA assaycan be used to detect and quantify purified His-tagged proteins.In addition, the limit of P78-His detection, based on threetimes the signal-to-noise level, was estimated to be about 4.2nM.To test the potential of this method for direct detection of

expressed recombinant proteins from cell lysate, differentamounts of P78-His, from 0 to 2 μg, were mixed with anindicated amount of E. coli cell lysate so that the total amountof protein in each sample was 20 μg. As shown in Figure 7, theproportional relationship between fluorescence increase (F/F0− 1) of HMBA and concentration of P78-His in E. coli lysatewas verified and used as the standard curve. Then, the plasmidcontaining P78-His gene was transformed into E. coli, andisopropyl-β-D-thiogalactopyranoside (IPTG) was used toinduce the expression of P78-His. After culture, the E. colicells were collected, and cell lysate was prepared. Western blotdata confirm the presence of P78-His in the cell lysate from theIPTG treated group, as well as the absence of P78-His in thecell lysate from the group without IPTG treatment (Figure 5B).Cell lysate (20 μg) from P78-His expressing E. coli with IPTGtreatment (F) or without IPTG treatment (F0) was tested,respectively, by HMBA, and the fluorescence recovery wasmeasured. The experiments were repeated three times, and theaverage value of fluorescence increase (F/F0 − 1) was

Figure 2. Fluorescence emission spectra under different conditions.From bottom to top: 800 nM P68-His only (green line); 25 nMHMBA (black line); 25 nM HMBA incubated with 800 nM P78-His(blue line); 25 nM HMBA incubated with its cDNA at a concentrationof 125 nM (red line). Excitation: 480 nm. Emission: 518 nm. Theincubation time was 30 min.

Figure 3. Fluorescence restoration of HMBA by P78-His or P78 as afunction of time. HMBA (25nM) was incubated in buffer for 6 min,and then, proteins were added. Excitation: 480 nm. Emission: 518 nm.

Figure 4. Fluorescence intensity changes (F/F0 − 1) of the HMBA(25nM) toward different proteins. F0 and F are fluorescence intensitiesat 520 nm in the absence or presence of detected proteins,respectively. Excitation: 480 nm; emission: 518 nm. Concentrationsof all proteins were 200 nM, except BSA*, whose concentration was 10μM. The concentration of peptide H6 was 8 μM.

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determined to be 0.441. According to the standard curve inFigure 7, the yield of expressed P78-His in E. coli lysate proteinswas determined to be 7.45%. Then, we calculated that theamount of expressed P78-His was 1.49 μg from the 20 μg celllysate proteins. To the best of our knowledge, this is the firstexample directly measuring the concentration and expressionyield of recombinant proteins from cell lysate, and the entireprocedure required only 5 min.In summary, this method utilized HMBA to target His-tag to

perform detection and quantitation of recombinant proteins.

Our design offers several advantages. First, because the HMBAassay can directly detect expressed recombinant proteins fromcell lysate, the entire procedure is much more rapid (only about5 min) than the traditional antibody-based affinity sensingstrategies requiring multiple reagents, incubations, and washingsteps. Second, this is a very simple and low cost method. TheHMBA itself is sensitive enough to detect the recombinantproteins from cell lysate. It does not require additionalmodifications, such as fixing the MBAs on the detectionsurface.15 Moreover, as a simple method, it is only necessary toadd protein samples to a solution containing HMBA and thenmeasure the fluorescence recovery to detect recombinantproteins. This kind of turn-on of fluorescence upon recognitionof oligohistidine was also reported by others using chemicalprobers,16 and we extended our design for direct detection ofrecombinant protein in cell lysates. Finally and moreimportantly, even though a few protein-specific aptamers havebeen selected and some of them were developed into associatedMBAs for protein detection,15,17 HMBA is superior to otherMBAs because it can target various different recombinantproteins, as long as they contain His-tags, while other MBAscan only be used to target one indicated protein. Therefore, thismethod could become a universal approach for the quantitativedetection of expressed recombinant proteins. Consequently,because our method is a rapid, low cost, universal, and simpleapproach, different laboratories from different research areassuch as molecular biology, biochemistry, and medicine caneasily use this design for the detection of various recombinantproteins. Our method holds great promise to become a routinetool for practical applications in recombinant protein detectionand will be very useful in the wide range of research areas ofrecombinant proteins.

■ EXPERIMENTAL SECTIONDNA Sequences. The underlined part is a major fragment

of 6H5 aptamer, which recognizes His-tag, and the bold partsform the HMBA stem.

Fluorescence Measurements. All fluorescence measure-ments were performed using a Fluorolog spectrophotometer(Jobin Yvon Horiba). The HMBA was prepared in PBS 1×without Ca and Mg (Dulbeco’s). The fluorescence spectra forall samples were measured at 20 °C.

His-Tag Protein Expression and Purification. Thehuman Pdx1 gene was cloned into pET28b vector. The full-length human GSTZ cDNA was cloned into pQE30 vector.The Rep68 or Rep78 of AAV2 was subcloned into pET-15b. Allproteins contained N-terminal His-tags. Soluble protein

Figure 5. Western blot analysis by anti-His-tag antibodies. (A) Analysis of purified His-tagged proteins; (B) Analysis of E. coli lysate: (1) lysate fromcells transformed by Rep78-His and induced by IPTG; (2) lysate from cells transformed by Rep78-His without treatment with IPTG.

Figure 6. Fluorescence emission spectra of HMBA (25 nM). (A) Withaddition of different concentrations of P78-His. From bottom to top:0, 6.25, 13, 25, 50, 100, 200, and 400 nM. (B) Linear relationshipbetween F/F0 and P78-His concentration. Data were obtained fromthe average of three parallel experiments.

Figure 7. Direct quantification of recombinant protein from cell lysate.HMBA (25nM) was incubated with 20 μg protein mixtures (purifiedP78-His plus E. coli lysate), in which the amount of P78-His variedfrom 1.25% to 10%. The proportional relationship betweenfluorescence increase of HMBA and concentration of P78-His in E.coli lysate was used as the standard curve. After that, fluorescenceemission spectra of HMBA were measured with addition of 20 μg ofcell lysate of E. coli expressing P78-His (F) or 20 μg of cell lysate of E.coli which did not express P78-His (F0). This F/F0 − 1 value (redspot) caused by P78-His expression was measured to be 0.441,corresponding to 7.45% of expression yield. Data were obtained fromthe average of three parallel experiments.

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expression was obtained in E. coli strain BL21 (DE3) by growthat 37 °C overnight, then 30 °C for 3 h, followed by induction ofIPTG with a final concentration of 1 mM at 18 °C for 24 h.Harvested cells were treated by three freeze/thaw cycles in 20mM Tris, pH 7.5, 0.5 M NaCl, 15% glycerol, and 10 mMimidazole buffer, followed by Benzonase Nuclease (Novagen)treatment. Soluble proteins were directly loaded onto aprepacked HisTrap column (GE) and eluted from the columnusing a gradient of imidazole from 10 to 400 mM. Fractionscontaining His-tagged proteins were combined and dialyzedagainst 20 mM Tris (pH7.5), 1 mM ethylenediaminetetraaceticacid (EDTA), 0.5 M NaCl, and 10% glycerol. Proteinconcentrations were measured by the Bradford method.Proteins of Rep 68 and Rep78 without His-tag were kindlyprovided by Dr. Nick Muzyczka, University of Florida.Cell Lysate Preparation. Harvested E. coli cells were

treated by repeated freeze/thaw cycles in 20 mM Tris, pH 7.5,0.5 M NaCl, and 15% glycerol. Samples were centrifuged at 14000g for 20 min to remove the cell debris. The cell lysate wasthen incubated with Benzonase Nuclease for 30 min at RT.EDTA (5 mM) was added to cell lysate, and finally, the totalprotein concentration was measured by the Bradford methodWestern Blot. The purified His-tagged proteins or cell

lysate was loaded into a sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) system. After electrophoresisand membrane transfer, the membrane was blocked, washed,and incubated with the anti-His-tag antibodies (1:2000dilution). Secondary antibodies (1:2000 dilution) were usedto visualize targeted proteins.

■ AUTHOR INFORMATION

Corresponding Author*E-mail: tan@chem.ufl.edu.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work was supported by the National Key ScientificProgram of China (2011CB911001, 2011CB911003) andChina National Instrumentation Program 2011YQ03012412.This work was also supported by grants awarded by theNational Institutes of Health (GM066137, GM079359, andCA133086).

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